WO2025127437A1 - Use of cyclo(his-pro) (chp) for enhancing exercise performance, improving muscle function, or preventing, alleviating, or treating muscle diseases - Google Patents

Abstract

The present invention relates to uses of cyclo(his-pro) (CHP) for enhancing exercise performance, improving muscle function, or preventing, alleviating, or treating muscle diseases and, more specifically, provides a pharmaceutical composition, a food composition, a feed additive, a method for enhancing exercise performance, a method for improving muscle function or for preventing, alleviating, or treating muscle diseases, and a method for enhancing muscle strength, each using cyclo(his-pro) as an active ingredient, wherein the cyclo(his-pro) can be used not only in enhancing exercise performance and strengthening muscle strength by exhibiting effects of increasing muscle strength, movement coordination, resistance to muscle fatigue, sense of balance, and muscle energy production, but also in enhancing muscle function or preventing, alleviating, or treating muscle diseases by exhibiting effects of increasing muscle mass and muscle fiber size, inhibiting muscle loss, muscle atrophy, and improving muscle energy production.

Description

Uses of Cyclo-Hyspro (CHP) for improving exercise performance, improving muscle function, or preventing, improving, or treating muscle diseases

This application claims priority to Republic of Korea Patent Application No. 10-2023-0183097, filed December 15, 2023, the entire contents of which are incorporated herein by reference.

The present invention relates to the use of cyclo-hispro (CHP) for enhancing exercise performance, improving muscle function, or preventing, improving, or treating muscle diseases, and more specifically, it exhibits the effects of increasing muscle strength, increasing movement coordination, increasing tolerance to muscle fatigue, increasing a sense of balance, and increasing muscle energy production, and thus can be used for enhancing exercise performance and strengthening muscle strength. In addition, it exhibits the effects of increasing muscle mass, increasing muscle fiber size, inhibiting muscle loss, inhibiting muscle atrophy, and increasing muscle energy production, and thus can be used for improving muscle function or preventing, improving, or treating muscle diseases, and provides a pharmaceutical composition, a food composition, a feed additive, and a method for enhancing exercise performance, a method for improving muscle function, or preventing, improving, or treating muscle diseases, and a method for strengthening muscle strength using the same.

Muscle atrophy is a progressive decrease in muscle mass, which causes muscle weakness and degeneration. Muscle atrophy is accelerated by inactivity, oxidative stress, or chronic inflammation, and weakens muscle function and motor skills.

One of the representative methods for improving muscle function by promoting energy consumption is to increase fatty acid oxidation by mitochondria to generate ATP energy. The number and ability of mitochondria that control this are regulated by the coactivator PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator 1 alpha), and it was revealed that PGC-1α activity is regulated by SIRT1 (sirtuin 1) (Non-patent Document 1).

When the transcription factor Fox (forhead box) moves from the cytoplasm to the nucleus, it increases the expression of the E3 ubiquitin ligase factors Atrogin-1/MAFbx (muscle atrophy F-box) and MuRF-1 (muscle RINGfinger protein-1), which are involved in protein degradation (Non-patent Document 2). When the expression levels of these factors increase, protein degradation within the muscle is promoted, resulting in a decrease in muscle mass. Therefore, suppression of the expression of Atrogin-1/MAFbx and MuRF-1 reduces the loss of muscle protein mass, thereby maintaining normal muscle function.

As the elderly population rapidly increases, research investments are being focused on developing treatments for geriatric diseases. In 2017, the World Health Organization (WHO) created sarcopenia related to skeletal muscle atrophy as a disease code (ICD-10-CM), and treatment development for it is actively underway. However, to date, there are no drugs approved by the US FDA for the treatment of sarcopenia.

Meanwhile, Cyclo-HisPro (CHP) is a naturally occurring circular dipeptide composed of histidine-proline, a metabolite of thyrotropin-releasing hormone (TRH), or a physiologically active dipeptide that is synthesized in the body through TRH metabolism and de novo, and is widely distributed throughout the brain, spinal cord, and gastrointestinal tract.

CHP has been reported to improve diabetes and to be effective in treating neurodegenerative diseases such as Alzheimer's. In addition, CHP has been reported to be effective in treating bone loss disease (Patent Document 1), but its effects in improving muscle function, enhancing exercise performance, and treating muscle diseases are unknown.

[Prior art literature]

[Patent Document]

(Patent Document 1) Republic of Korea Publication Patent No. 10-2019-0058346

[Non-patent literature]

(Non-patent Document 1) Gerhart-Hines Z, Rodgers JT, Bare O, Lerin C, Kim SH, Mostoslavsky R, Alt FW, Wu Z, Puigserver P. Metabolic control of muscle mitochondrial function and fatty acid oxidation through SIRT1/PGC-1alpha. EMBO J. 2007 Apr 4;26(7):1913-23.

(Non-patent Document 2) Bonaldo P, Sandri M. Cellular and molecular mechanisms of muscle atrophy. Dis Model Mech. 2013 Jan;6(1):25-39.

The purpose of the present invention is to provide a composition for improving exercise performance containing cyclo-hispro as an active ingredient.

Another object of the present invention is to provide a composition for improving muscle function or preventing, improving or treating muscle disease, comprising cyclo-hispro as an active ingredient.

Another object of the present invention is to provide a composition for strengthening muscle, comprising cyclo-hispro as an effective ingredient.

Another object of the present invention is to provide a method for improving exercise performance, a method for improving muscle function, a method for preventing, improving or treating muscle disease and a method for strengthening muscle strength using cyclo-hispro.

Another object of the present invention is to provide a use of cyclo-hispro for the manufacture of a drug or health functional food for improving exercise performance, improving muscle function, preventing, improving or treating muscle diseases and strengthening muscle strength.

To solve the above-described problems, the present invention provides a pharmaceutical composition for improving exercise performance comprising cyclo-hispro or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a health functional food composition for improving exercise performance comprising cyclo-hispro or a food-wise acceptable salt thereof.

In addition, the present invention provides a feed additive for improving exercise performance comprising cyclo-hispro or a salt thereof.

In addition, the present invention provides a method for improving exercise performance, comprising administering an effective amount of cyclo-hispro or a salt thereof to a subject in need thereof.

In addition, the present invention provides a use of cyclo-hispro or a salt thereof for the manufacture of a drug for improving exercise performance or a health functional food.

In the present invention, the cyclo-hispro or a salt thereof can exhibit one or more of the following effects:

i) Increased muscle strength;

ii) Increased motor coordination;

iv) increased sense of balance; and

v) Increased muscle energy production.

Additionally, the present invention provides a pharmaceutical composition for improving muscle function or preventing or treating muscle disease, comprising cyclo-hispro or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a health functional food composition for improving muscle function or preventing or improving muscle disease, comprising cyclo-hispro or a food-wise acceptable salt thereof.

In addition, the present invention provides a feed additive for improving muscle function or preventing or improving muscle disease, comprising cyclo-hispro or a salt thereof.

Furthermore, the present invention provides a method for improving muscle function or preventing, improving or treating muscle disease, comprising administering an effective amount of cyclo-hispro or a salt thereof to a subject in need thereof.

In addition, the present invention provides the use of cyclo-hispro or a salt thereof for the manufacture of a medicament or health functional food for improving muscle function or preventing, improving or treating muscle disease.

In the present invention, the muscle disease may be a muscle disease caused by muscle dysfunction, muscle atrophy, muscle wasting, or muscle degeneration.

In the present invention, the muscle disease may be at least one selected from the group consisting of atony, muscular atrophy, muscular dystrophy, myasthenia, cachexia, sarcopenia, myocardia, and acardiotrophy.

In the present invention, the cyclo-hispro or a salt thereof can exhibit one or more of the following effects:

i) Increased muscle strength;

ii) Increased motor coordination;

iii) Increased tolerance to muscle fatigue;

iv) increased sense of balance; and

v) Increased muscle energy production.

Furthermore, the present invention provides a muscle strengthening composition comprising cyclo-hispro or a pharmaceutically or food-wise acceptable salt thereof.

The present invention also provides a method for strengthening muscle, comprising administering an effective amount of cyclo-hispro or a salt thereof to a subject in need thereof.

In addition, the present invention provides a use of cyclo-hispro or a salt thereof for the manufacture of a muscle strengthening agent or a health functional food.

The composition comprising cyclo-hispro of the present invention exhibits effects of increasing muscle strength, increasing movement coordination, increasing resistance to muscle fatigue, increasing a sense of balance, and increasing muscle energy production, and thus can be utilized as a medicine, health functional food, or feed additive for improving exercise performance or strengthening muscle strength. In addition, the composition exhibits effects of increasing muscle mass, increasing muscle fiber size, inhibiting muscle atrophy, inhibiting muscle loss, and increasing muscle energy production, and thus can be utilized as a medicine, health functional food, or feed additive for improving muscle function or preventing, improving, or treating various muscle diseases.

Figure 1 shows the survival rate of mice in each group during the experimental period.

Figures 2a to 2c show the results of confirming the effect of CHP administration on improving muscle function and enhancing exercise performance in an aged animal model. Figure 2a shows the results of grip strength measurement, Figure 2b shows the results of a limb hanging test, and Figure 2c shows the results of a sensorimotor function evaluation.

Figure 3a shows the changes in the weight of gastrocnemius (GM), tibialis anterior (TA), and cardiac muscle tissues following CHP administration in aged animals.

Figures 3b to 3d show changes in the cross-sectional area of muscle fibers according to CHP administration in an aged animal model. Figure 3b is a photograph of a cross-section of muscle fibers from GM muscles stained with H&E, Figure 3c shows the average cross-sectional area of muscle fibers measured, and Figure 3d shows the distribution of muscle fibers by cross-sectional area size as a percentage.

Figure 4 shows the changes in gene expression in GM and TA muscles following CHP administration in an aged animal model.

Figure 5 shows the change in citrate synthase activity in TA muscle following CHP administration in an aged animal model.

Figure 6 shows changes in gene expression related to cardiac atrophy following CHP administration in an aged animal model.

Figure 7a shows the results of grip strength measurements according to the CHP prevention protocol in the mdx animal model.

Figure 7b shows the results of grip strength measurements according to the CHP treatment protocol in the mdx animal model.

Figure 8 shows the results of the hanging test according to the CHP treatment protocol in the mdx animal model.

Figures 9a to 9f show the results of confirming the effect of maintaining force generation according to the CHP prevention protocol in the mdx animal model. Figure 9a shows the force-frequency relationship of the extensor digitorum longus (EDL), Figure 9b shows the maximum specific isometric force of the EDL developed by 25 Hz stimulation, Figure 9c shows the maximum specific isometric force of the EDL developed during the test, Figure 9d shows the force-frequency relationship of the soleus muscles, Figure 9e shows the maximum specific isometric force of the soleus muscle developed by 25 Hz stimulation, and Figure 9f shows the maximum specific isometric force of the soleus muscle developed during the test.

Figures 10a to 10d show the effects of CHP on Ca2 ⁺ dysregulation according to the CHP prevention protocol in the mdx animal model. Figure 10a shows Ca2 ⁺ amplitude (SR store) as a percentage of response in fibers isolated from BL10 mice upon 2.5 mM caffeine stimulation (n=10-12), Figure 10b shows SR Ca2 ⁺ uptake as a percentage of Ca2 ⁺ peak after 2.5 mM caffeine stimulation (n=10-12), Figure 10c shows Ca2 ⁺ amplitude (SR store) as a percentage of response in fibers isolated from BL10 mice upon 1 μM thapsigargin stimulation (n=6-14), and Figure 10d shows SR Ca2 ⁺ uptake as a percentage of Ca2 ⁺ peak after 1 μM thapsigargin stimulation (n=6-14).

Hereinafter, the present invention will be described in more detail.

As described above, with the rapid increase in the elderly population, active development of treatments for sarcopenia related to skeletal muscle atrophy is underway, but to date, there are no drugs that have shown a clear therapeutic effect.

Accordingly, the inventors of the present invention have confirmed that cyclo-hispro not only significantly enhances muscle strength, motor coordination, resistance to muscle fatigue, sense of balance, and muscle energy production, but also suppresses the expression of various genes related to aging and muscle atrophy and promotes the expression of genes related to anti-aging and anti-oxidation including mitochondrial function, thereby exhibiting excellent effects in improving exercise performance, strengthening muscle strength, improving muscle function, and preventing, improving, or treating muscle diseases, thereby completing the present invention.

Accordingly, the first aspect of the present invention relates to a composition for improving exercise performance comprising cyclo-hispro or a salt thereof, consisting essentially of said cyclo-hispro or a salt thereof, or consisting of said cyclo-hispro or a salt thereof.

Specifically, the composition comprises cyclo-hispro or a pharmaceutically acceptable salt thereof, or consists essentially of, or consists of cyclo-hispro or a pharmaceutically acceptable salt thereof; a pharmaceutical composition for enhancing exercise performance comprising cyclo-hispro or a food science-based acceptable salt thereof, or consists essentially of, or consists of cyclo-hispro or a food science-based acceptable salt thereof; Or it may be a feed additive for improving exercise performance comprising cyclo-hispro or a salt thereof, consisting essentially of said cyclo-hispro or a salt thereof, or consisting of said cyclo-hispro or a salt thereof.

In relation to the first aspect, the present invention provides a method for improving exercise performance, comprising administering an effective amount of cyclo-hispro or a pharmaceutically or food-wise acceptable salt thereof to a subject in need thereof.

In connection with the first aspect, the present invention also provides the use of cyclo-hispro or a pharmaceutically or food-wise acceptable salt thereof for improving exercise performance.

In relation to the first aspect, the present invention also provides the use of cyclo-hispro or a pharmaceutically or foodologically acceptable salt thereof for the manufacture of a drug or health functional food for improving exercise performance.

In the present invention, the cyclo-hisp may be used synthetically or as a commercially available product. In addition, it may be used by purifying it from a substance containing cyclo-hisp, such as a prostate extract or soybean hydrolysate.

By the use of the term "purified", it is meant that Cyclo-Hyspro is in a concentrated form compared to the form in which it can be obtained from natural sources, such as prostate extract. Purified ingredients can be concentrated from their natural sources, or obtained through chemical synthesis methods.

In the present invention, the term "exercise performance ability" means the ability to perform physical movements performed in daily life or sports quickly, strongly, for a long time, and skillfully. Exercise performance ability is defined by factors such as muscle strength, sense of balance, motor coordination, agility, and endurance. In addition, improving exercise performance ability in the present invention is considered to be a beneficial physiological effect even in people performing daily physical activities not related to exercise.

The term "muscle fatigue" in the present invention means a state in which the ability to perform physical activity is temporarily reduced after intense exercise or due to long-term exercise, and is accompanied by a decrease in muscle contractility, etc. Muscle fatigue may manifest itself as symptoms of fatigue, decreased endurance, decreased explosiveness, or lethargy.

In the present invention, the term "endurance" is defined as resistance to fatigue. It means resistance to fatigue that occurs during submaximal (before exerting maximum effort) sustained exercise or intense exercise. Endurance exercise usually lasts for 30 minutes or more. In particular, exercise that lasts 4 to 5 hours or more is also called ultra-endurance exercise. In addition, in the present invention, the increase in endurance is considered to be a beneficial physiological function when performing exercise that is not limited in time (such as recreational running, walking, swimming, cycling, and gymnastic training).

In a specific embodiment of the present invention, the effect of cyclo-hispro on improving motor performance was evaluated by performing grip strength measurement, limb hanging test, and sensorimotor function evaluation on an aged animal model treated with cyclo-hispro.

As a result, as shown in FIGS. 2a to 2c, the cyclo-hispro administered group significantly increased maximum muscle strength and hanging time, and significantly improved sensorimotor functions compared to the vehicle-treated control group, indicating effects of enhancing muscle strength, motor coordination, tolerance to muscle fatigue, and sense of balance. Accordingly, the cyclo-hispro according to the present invention can be utilized in various ways as a medicine, health functional food, feed additive, etc. for the purpose of improving exercise performance ability.

In addition, cyclo-hispro or a salt thereof according to the present invention can be administered to a subject for the purpose of improving exercise performance ability. Accordingly, a method for improving exercise performance ability is provided, comprising a step of administering cyclo-hispro or a salt thereof to a subject in need thereof.

The second aspect of the present invention relates to a composition for improving muscle function or preventing, improving or treating muscle disease, comprising cyclo-hispro or a salt thereof, consisting essentially of said cyclo-hispro or a salt thereof, or consisting of said cyclo-hispro or a salt thereof.

Specifically, the composition comprises cyclo-hispro or a pharmaceutically acceptable salt thereof, or consists essentially of, or consists of cyclo-hispro or a pharmaceutically acceptable salt thereof, a pharmaceutical composition for improving muscle function or preventing or treating muscle disease; a health functional food composition for improving muscle function or preventing or improving muscle disease, comprising cyclo-hispro or a food acceptable salt thereof, or consisting essentially of, or consisting of cyclo-hispro or a food acceptable salt thereof. Or it may be a feed additive for improving muscle function or preventing or improving muscle disease, comprising cyclo-hispro or a salt thereof, consisting essentially of said cyclo-hispro or a salt thereof, or consisting of said cyclo-hispro or a salt thereof.

In relation to the second aspect, the present invention provides a method for improving muscle function or preventing, improving or treating muscle disease, comprising administering an effective amount of cyclo-hispro or a pharmaceutically or food-wise acceptable salt thereof to a subject in need thereof.

In connection with the second aspect, the present invention also provides the use of cyclo-hispro or a pharmaceutically or food-wise acceptable salt thereof for improving muscle function or preventing, improving or treating muscle diseases.

In relation to the second aspect, the present invention also provides the use of cyclo-hispro or a pharmaceutically or foodologically acceptable salt thereof for the manufacture of a medicament or health functional food for improving muscle function or preventing, improving or treating muscle disease.

In the present invention, the description of cyclo-hispro used as an effective ingredient is the same as that described in the first aspect, and therefore, its description is omitted.

In the present invention, the muscle disease is preferably a disease reported in the art as a muscle disease caused by muscle dysfunction, muscle atrophy, muscle wasting or muscle degeneration, and may be at least one selected from the group consisting of atony, muscular atrophy, muscular dystrophy, myasthenia, cachexia, sarcopenia, myocardia and acardiotrophy, but is not limited thereto.

The above muscle dysfunction, muscle atrophy, muscle wasting or muscle degeneration may be caused by genetic factors, acquired factors, diseases causing muscle loss or weakening, or aging, and according to one embodiment, may be caused by aging. Alternatively, the muscle dysfunction, muscle atrophy, muscle wasting or muscle degeneration may be a side effect of disease treatment, for example, a side effect of anticancer treatment. The muscle wasting is characterized by a gradual loss of muscle mass, and weakening and degeneration of muscles, particularly skeletal muscle or voluntary muscle and cardiac muscle. In particular, in the present invention, the "muscle disease" may be a disease in which muscle dysfunction, muscle atrophy, muscle wasting or muscle degeneration occurs due to aging of the muscle itself, for example, skeletal muscle or cardiac muscle itself, regardless of the death of motor neurons in the central nervous system, or a disease in which muscle dysfunction, muscle atrophy, muscle wasting or muscle degeneration occurs due to a genetic mutation.

As used herein, “old age” or “aging” is generally defined as a noticeable decline or loss of muscle mass beginning around age 50, with sarcopenia (age-related muscle loss) becoming more pronounced around age 60.

In a specific embodiment of the present invention, changes in muscle weight and muscle fiber size were confirmed in an aged animal model treated with cyclo-hispro.

As a result, as shown in FIGS. 3a to 3d, it was confirmed that the cyclo-hispro administered group had an increase in the muscle weight of the tibialis anterior (TA) muscle and an increase in the average cross-sectional area of muscle fibers compared to the control group treated with the vehicle. Therefore, the cyclo-hispro according to the present invention can be effectively used for improving muscle function through an increase in muscle mass and muscle fiber size, as well as preventing, improving, or treating muscle diseases caused by decreased muscle function, muscle atrophy, muscle wasting, or muscle degeneration.

E3 ubiquitin ligase factors Atrogin-1/MAFbx (Muscle atrophy F-box) and MuRF-1 (Muscle RING-finger protein-1) are involved in protein degradation, and when their expression levels increase, protein degradation in muscles is promoted, resulting in a decrease in muscle mass.

PGC-1α is an important factor that regulates energy metabolism in muscles, regulating fatty acid oxidation and increasing energy levels by promoting mitochondrial biogenesis. In addition, PGC-1α is known to activate transcription factors that affect mitochondrial proliferation, energy homeostasis regulation, and respiration, such as NRF1, TFAM, and Sirt-1.

In addition, genes related to aging and muscle atrophy include Myostatin, Foxo-1, Dystrophin, Sirt-3, Nrf-2, Known genes involved in mitochondrial biogenesis include Err-α, Cox-4, Drp-1, Tf1bm, and Tf2bm.

Accordingly, in a specific embodiment of the present invention, the expression levels of the genes presented in Table 2 were confirmed in an aged animal model treated with cyclo-hispro, thereby evaluating the effect of cyclo-hispro on improving muscle function and preventing, improving or treating muscle disease.

As a result, as shown in Fig. 4, the cyclo-hispro-administered group showed a significant decrease in the gene expression levels of Myostatin, Atrogin-1, Foxo-1, and Dystrophin in skeletal muscle compared to the vehicle-treated control group, confirming that cyclo-hispro can be effectively used for the prevention, improvement, or treatment of muscle diseases caused by decreased muscle function, muscle atrophy, muscle wasting, or muscle degeneration through its muscle wasting inhibition and muscle atrophy inhibition activities.

In addition, as shown in Fig. 4, it was confirmed that the gene expression levels of PGC-1α, Nrf-2, Sirt-1, Sirt-3, Drp-1, Err-α, Cox-4, Tf1bm and Tf2bm in skeletal muscle were significantly increased in the cyclo-hispro administered group compared to the vehicle-treated control group. Therefore, cyclo-hispro according to the present invention helps muscle energy production by increasing the expression of mitochondrial-mediated energy metabolism regulators and suppresses muscle atrophy, thereby improving muscle function as well as treating muscle diseases caused by decreased muscle function, muscle atrophy, muscle wasting or muscle degeneration.

Oxidation-related enzymes of the Krebs cycle (or TCA cycle) are used as biomarkers related to energy production and supply efficiency. Citrate synthase is an enzyme that catalyzes citrate synthesis in the first step of the TCA cycle. If the TCA cycle does not run smoothly, blood lactate accumulates, causing fatigue.

Accordingly, in a specific embodiment of the present invention, the activity change of citrate synthase was confirmed in an aged animal model treated with cyclo-hispro, thereby evaluating the effects of cyclo-hispro on improving exercise performance, muscle function, and preventing, improving, or treating muscle diseases.

As a result, as shown in Fig. 5, the cyclo-hispro administration group was confirmed to be effective in improving exercise performance, muscle function, and preventing, improving, or treating muscle diseases by increasing the activity of citrate synthase in skeletal muscle compared to the vehicle-treated control group, thereby promoting recovery from muscle fatigue and increasing energy production and supply efficiency.

In another specific embodiment of the present invention, to evaluate whether cyclo-hispro can inhibit muscle loss and muscle atrophy of not only skeletal muscle but also cardiac muscle, the gene expression levels of Atrogin-1 and Murf-1 were determined in cardiac muscle of an aged animal model treated with cyclo-hispro.

As a result, as shown in Fig. 6, the cyclo-hispro administration group significantly reduced the gene expression levels of Atrogin-1 and Murf-1 in the myocardium compared to the vehicle-treated control group, thereby confirming that it can be effectively used for the prevention, improvement, or treatment of diseases caused by myocardial abnormalities through the inhibition of myocardial muscle loss and myocardial muscle atrophy inhibition activities.

In the present invention, the disease occurring due to an abnormality in the heart muscle may be, for example, cardiomyopathy or cardiac atrophy occurring primarily in the heart muscle itself, but is not limited thereto.

Acardiotrophy is caused by starvation, wasting disease (cancer, etc.), and aging, and the myocardial fibers become thin and thin, and the nuclei become condensed and fixed in size. Accordingly, the muscle fascicles also decrease in volume, the entire heart becomes smaller, the subepicardial adipose tissue is markedly reduced, and the coronary arteries become curved. A brown pigment called a wasting pigment (lipofuscine) appears at both ends of the nuclei of the myocardial fibers, and with the reduction of adipose tissue, the entire heart takes on a brownish tone.

The most common muscular dystrophy is X-linked Duchenne/Becker muscular dystrophy, but there are also several other types, including limb-girdling, fascioscapulohumeral, rigid, and Fukuyama, depending on the type of protein that makes up the muscle membrane.

Duchenne muscular dystrophy (DMD) is one of the most devastating and progressive inherited muscular dystrophies that appears in childhood. It is caused by mutations in the dystrophin gene and is a recessive X-linked genetic disorder that affects 1 in 3,500 male births. Notably, female carriers can also develop the disease. Dystrophin is a key member of the dystrophin-associated protein complex (DAPC), the basic link between the cytoskeleton and the extracellular matrix (ECM) of muscle fibers. Without functional dystrophin, DAPC is degraded, resulting in the loss of stable cytoskeleton-ECM connections, weakening the sarcolemma and predisposing muscle fibers to contractile damage.

The first clinical signs appear in childhood, between the ages of 2 and 3, and children show motor developmental delays, with difficulty walking or jumping. Dystrophy first affects the proximal muscles and then spreads to the distal limb muscles, and children become wheelchair-bound by the age of 12. Dystrophy eventually reaches the respiratory muscles, requiring artificial ventilation. The heart muscle is also weakened, ultimately resulting in cardiomyopathy. In fact, respiratory and heart failure are the two main causes of death in DMD.

Despite all the therapeutic advances over the past several decades, there is still no approved cure for DMD. Nevertheless, improvements in treatment protocols and symptom management can delay the progression of DMD and improve the quality of life of patients. With proper treatment, the life expectancy of DMD patients today is approximately 28 years. The current standard of care is corticosteroid therapy, which reduces chronic inflammation and delays muscle damage caused by inflammation. In particular, long-term use of corticosteroids has several side effects, including weight gain, growth retardation, and osteoporosis.

The most widely used animal model of DMD is the Dmdmdx (mdx) mouse. This model, generated on a C57BL/10ScSn background, carries a nonsense mutation in exon 23 of the dystrophin gene, creating an early stop codon and thus a truncated form of the protein.

In a specific embodiment of the present invention, the effect of cyclo-hispro on improving exercise performance was evaluated by performing grip strength measurement and limb hanging test according to the cyclo-hispro prevention and treatment protocol in an mdx mouse model. As a result, as shown in Figures 7a, 7b, and 8, it was confirmed that the cyclo-hispro administration group significantly increased grip strength and hanging time compared to the mdx control group treated with water, showing the effect of increasing limb muscle strength and exercise endurance.

In another specific embodiment of the present invention, the contractile and force production effects were evaluated in extensor digitorum longus (EDL) and soleus muscles isolated from an mdx mouse model to which a cyclo-hispro prophylaxis protocol was applied. As a result, as shown in FIGS. 9A to 9F , the muscles of the mdx mouse showed overall lower force production and a downward shift in the force-frequency relationship compared to the BL10 mouse. The performance of the EDL and soleus muscles was improved by CHP, and the maximal contractile force was partially restored. This suggests that the known cyclo-hispro treatment maintains force production.

In another specific embodiment of the present invention, the effect of cyclo-hispro on Ca2 ⁺ dysregulation was evaluated by evaluating the recovery of Ca2 ⁺ release/uptake from the sarcoplasmic reticulum (SR) of flexor digitorum brevis (FDB) muscle isolated from the mdx mouse model to which the cyclo-hispro preventive protocol was applied. As a result, as shown in Fig. 10a, the response to caffeine-induced Ca2 ⁺ release in the SR was recovered by cyclo-hispro, and as shown in Fig. 10b, although Ca2 ⁺ uptake through store-operated calcium channels (SOC) after SR depletion is generally upregulated in mdx as a compensatory mechanism for Ca2 ⁺ depletion, CHP normalized Ca2 ⁺ uptake, thereby maintaining appropriate Ca2 ⁺ handling. Additionally, to directly quantify SOCE, stimulation of SR Ca2 ⁺ release was performed using thapsigargin instead of caffeine, and a similar trend of cyclo-hispro-induced normalization was confirmed, as shown in Figures 10c and 10d.

In addition, cyclo-hispro or a salt thereof according to the present invention can be administered to a subject for the purpose of improving muscle function or preventing, improving or treating muscle disease. Accordingly, a method for improving muscle function or preventing, improving or treating muscle disease is provided, comprising a step of administering cyclo-hispro or a salt thereof to a subject in need thereof.

A third aspect of the present invention relates to a composition for muscle strengthening comprising cyclo-hispro or a salt thereof, consisting essentially of said cyclo-hispro or a salt thereof, or consisting of said cyclo-hispro or a salt thereof.

Specifically, the composition comprises a pharmaceutical composition for muscle strengthening comprising cyclo-hispro or a pharmaceutically acceptable salt thereof, consisting essentially of, or consisting of cyclo-hispro or a pharmaceutically acceptable salt thereof; a health functional food composition for muscle strengthening comprising cyclo-hispro or a food acceptable salt thereof, consisting essentially of, or consisting of cyclo-hispro or a food acceptable salt thereof; Or it may be a muscle strengthening feed additive comprising cyclo-hispro or a salt thereof, consisting essentially of said cyclo-hispro or a salt thereof, or consisting of said cyclo-hispro or a salt thereof.

In relation to the third aspect, the present invention provides a method for strengthening muscle, comprising administering to a subject in need thereof an effective amount of cyclo-hispro or a pharmaceutically or food-wise acceptable salt thereof.

In connection with the third aspect, the present invention also provides the use of cyclo-hispro or a pharmaceutically or foodologically acceptable salt thereof for muscle strengthening.

In connection with the third aspect, the present invention also provides the use of cyclo-hispro or a pharmaceutically or foodologically acceptable salt thereof for the manufacture of a muscle strengthening agent or a health functional food.

In the present invention, the description of cyclo-hispro used as an effective ingredient is the same as that described in the first aspect, and therefore, its description is omitted.

The term "strength enhancement" of the present invention refers to the effects of enhancing physical performance, enhancing maximum endurance, increasing muscle mass, enhancing muscle recovery, reducing muscle fatigue, improving energy balance, or a combination thereof.

Cyclo-hispro or a salt thereof according to the present invention not only exhibits the effects of increasing muscle strength, increasing movement coordination, increasing resistance to muscle fatigue, and increasing a sense of balance as described in the first and second aspects, but also exhibits the effects of increasing muscle mass, increasing muscle fiber size, inhibiting muscle atrophy, inhibiting muscle loss, and increasing muscle energy production, and thus can be utilized in various ways as a pharmaceutical product, a health functional food product, and a feed additive for the purpose of strengthening muscle strength.

In addition, cyclo-hispro or a salt thereof according to the present invention can be administered to a subject for the purpose of strengthening muscle strength. Accordingly, a method for strengthening muscle strength is provided, comprising a step of administering cyclo-hispro or a salt thereof to a subject in need thereof.

The term "prevention" as used in the present invention means any act of inhibiting or delaying the onset of a muscle disease by administering a composition according to the present invention.

The term "improvement" as used in the present invention means any action that at least reduces a parameter related to the condition being treated, for example, the degree of symptoms.

The term "treatment" as used in the present invention means any action by which symptoms of a muscle disease are improved, symptoms are prevented from worsening, or symptoms are beneficially changed by administration of a composition according to the present invention.

The term "increase," as used herein, means, for example, an increase in the expression of a specific biomarker gene or protein, which is about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more higher, compared to a control group not administered the active ingredient or a composition comprising, consisting essentially of, or consisting of the active ingredient of the present invention under the same conditions.

The term "decrease or reduction" or "suppression" as used herein, for example, a decrease or suppression of the expression of a specific biomarker gene or protein, means a decrease of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, when compared to a control group not administered the active ingredient or the composition comprising, consisting essentially of, or consisting of the active ingredient of the present invention, under the same conditions.

The term "improvement" as used in the present invention, for example, improvement in exercise performance or improvement in muscle function, means an increase of about 5% or more, about 10% or more, about 15% or more, about 20% or more, when compared to a control group not administered the active ingredient or the composition comprising, consisting essentially of, or consisting of the active ingredient of the present invention under the same conditions.

In the present invention, the term "pharmaceutically acceptable" means physiologically acceptable and does not typically cause an allergic reaction or similar reaction when administered to a human, and the salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid.

The pharmaceutically acceptable salt may be an acid addition salt formed using an organic acid or an inorganic acid, wherein the organic acid includes, for example, formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid, malonic acid, fumaric acid, succinic acid, succinic acid monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxy acetic acid, benzenesulfonic acid, p-toluenesulfonic acid or methanesulfonic acid. The inorganic acid includes, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid or boric acid. The acid addition salt may preferably be in the form of a hydrochloride or an acetate salt, more preferably in the form of a hydrochloride salt.

In addition, additional possible salt forms include gabapentin salt, gabapentin salt, pregabalin salt, nicotinate salt, adipate salt, hemimalonate salt, cysteine salt, acetylcysteine salt, methionine salt, arginine salt, lysine salt, ornithine salt, or aspartate salt, etc.

In addition, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may further comprise, for example, a carrier for oral administration or a carrier for parenteral administration. The carrier for oral administration may comprise lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. The carrier for parenteral administration may comprise water, suitable oils, saline, aqueous glucose and glycols, and the like. In addition, the composition may further comprise a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally, and parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration.

The pharmaceutical composition of the present invention can be formulated as a preparation for oral administration or parenteral administration according to the administration route as described above. When formulated, it can be prepared using one or more buffers (e.g., saline or PBS), carbohydrates (e.g., glucose, mannose, sucrose, or dextran, etc.), antioxidants, bacteriostats, chelating agents (e.g., EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrating agents, or surfactants, diluents, or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions or capsules, and these solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient, for example, starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethyl cellulose, and hydroxypropyl methyl-cellulose or gelatin. For example, a tablet or a sugar-coated tablet can be obtained by mixing an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing it into a granule mixture.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, or syrups, and may contain various excipients such as wetting agents, sweeteners, flavoring agents, or preservatives in addition to the commonly used simple diluents such as water or liquid paraffin.

In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrating agent depending on the case, and anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be additionally included.

When administered parenterally, the pharmaceutical composition of the present invention may be formulated in the form of injections, transdermal administration agents, and nasal inhalers together with a suitable parenteral carrier according to methods known in the art. In the case of the injection, it must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injection include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and/or vegetable oils. More preferably, suitable carriers include Hanks' solution, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. In order to protect the above injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc. may be additionally included. In addition, the above injection may in most cases additionally include an isotonic agent such as sugar or sodium chloride.

In the case of percutaneous administration, the forms include ointments, creams, lotions, gels, external solutions, pastes, liniments, aerosols, etc. As used above, 'percutaneous administration' means topically administering a pharmaceutical composition to the skin so that an effective amount of active ingredients contained in the pharmaceutical composition are delivered into the skin.

For inhalation administration, the compounds used according to the invention may conveniently be delivered in the form of an aerosol spray from a pressurized pack or nebulizer, using a suitable propellant, for example, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve which delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in the well-known prescription book of pharmaceutical chemistry (Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour).

The pharmaceutical composition of the present invention can provide a desirable effect of improving exercise performance, improving muscle function, and/or preventing, improving, or treating muscle disease when it contains an effective amount of cyclo-hispro or a pharmaceutically acceptable salt thereof. As used herein, the term "effective amount" refers to an amount that shows a greater response than a negative control, and preferably refers to an amount sufficient to improve exercise performance, improve muscle function, and/or prevent, improve, or treat muscle disease. The pharmaceutical composition of the present invention may contain 0.01 to 99.9% of cyclo-hispro or a pharmaceutically acceptable salt thereof based on the total amount of the composition, and the remainder may be comprised of a pharmaceutically acceptable carrier. The effective amount of cyclo-hispro or a pharmaceutically acceptable salt thereof contained in the pharmaceutical composition of the present invention will vary depending on the form in which the composition is commercialized, etc.

The total effective amount of the pharmaceutical composition of the present invention can be administered to a patient as a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention can vary the content of the effective ingredient depending on the condition of the patient. For example, based on cyclo-hispro or a pharmaceutically acceptable salt thereof, it can be administered in one to several divided doses, preferably in an amount of 0.001 to 100 mg, more preferably 0.01 to 10 mg per kg of body weight per day. However, since the dosage of the cyclo-hispro or a pharmaceutically acceptable salt thereof is determined as an effective dosage for a patient by taking into consideration various factors such as the route of administration and the number of treatments of the pharmaceutical composition as well as the patient's age, weight, health condition, sex, severity of disease, diet, and excretion rate, taking these points into consideration, a person having ordinary skill in the art will be able to determine an appropriate effective dosage of the cyclo-hispro or a pharmaceutically acceptable salt thereof for a specific use for enhancing exercise performance, improving muscle function, and/or preventing, improving, or treating muscle disease. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.

In the present invention, the term "food-wise acceptable" means physiologically acceptable and does not typically cause an allergic reaction or similar reaction when ingested by humans, and the salt is preferably an acid addition salt formed by a food-wise acceptable free acid.

In the present invention, preferred examples of “food-related acceptable salts” may include the types of “pharmaceutically acceptable salts” described above.

In the present invention, the term “health functional food” includes both the meanings of “functional food” and “health food”.

In the present invention, the term "functional food" is the same as food for special health use (FoSHU), and means a food with high medical and healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition.

In the present invention, the term "health food" means a food that has a more active health maintenance or promotion effect than general food, and health supplement food means a food for the purpose of health supplementation. In some cases, the terms functional food, health food, and health supplement food are used interchangeably. The food can be manufactured in various forms such as tablets, capsules, powders, granules, liquids, and pills.

As a specific example of such functional foods, processed foods can be manufactured by using the composition to transform and preserve the characteristics of agricultural, livestock or marine products while improving their storage properties.

The health functional food composition of the present invention can also be manufactured in the form of nutritional supplements or dietary supplements, food additives, etc., and is intended for human consumption.

The food composition of the above type can be manufactured in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and processed foods thereof (e.g., ham, sausages, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juices, various drinks, cookies, taffy, dairy products (e.g., butter, cheese, etc.), edible plant fats, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.), etc., which can be manufactured by adding cyclo-hyspro or a food scientifically acceptable salt thereof.

In addition, nutritional supplements may be manufactured by adding cyclo-hispro or a food-chemically acceptable salt thereof to capsules, tablets, pills, etc., but are not limited thereto.

In addition, as a health functional food, although not limited thereto, for example, the cyclo-hispro or a food scientifically acceptable salt thereof can be manufactured in the form of tea, juice, and drink and consumed (health beverage) by liquefying, granulating, encapsulating, and powdering. In addition, in order to use the cyclo-hispro or a food scientifically acceptable salt thereof in the form of a food additive, it can be manufactured in the form of a powder or concentrate and used. In addition, the cyclo-hispro or a food scientifically acceptable salt thereof can be manufactured in the form of a composition by mixing with a known active ingredient known to be effective in improving exercise performance, improving muscle function, and/or preventing, improving, or treating muscle diseases.

When the food composition of the present invention is used as a health beverage composition, the health beverage composition may contain various flavoring agents or natural carbohydrates as additional ingredients, like a typical beverage. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol and erythritol. The sweetener may be a natural sweetener such as thaumatin and stevia extract; a synthetic sweetener such as saccharin and aspartame, etc. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention.

Cyclo-hispro or a food-chemically acceptable salt thereof may be contained as an effective ingredient in a food composition for improving exercise performance, improving muscle function, and/or preventing or improving muscle disease, and the amount thereof is an amount effective to obtain the above effects, and is preferably, for example, 0.01 to 100 wt% based on the total weight of the entire composition, but is not particularly limited thereto.

In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, or carbonating agents. In addition, the health functional food of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in mixtures. The ratio of these additives is not particularly important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In the present invention, the term "feed" means a substance that supplies organic or inorganic nutrients necessary for sustaining the life of an animal. The feed includes nutrients such as energy, protein, lipid, vitamins, and minerals required by animals such as livestock, and may be plant-based feed such as grains, roots, fruits, food processing by-products, algae, fibers, fats, starches, meal, and grain by-products, or animal-based feed such as proteins, inorganic substances, fats, minerals, fats, and single-cell proteins, but is not limited thereto.

In the present invention, "feed additive" means a substance added to feed to improve animal productivity or health, and is not particularly limited thereto, but may additionally include amino acids, vitamins, enzymes, flavoring agents, silicates, buffers, extractants, oligosaccharides, etc. for growth promotion, disease prevention, etc.

The content of cyclo-hispro or a salt thereof included in the feed additive of the present invention is not particularly limited, but may be, for example, 0.001 to 1 % (w/w), 0.005 to 0.9 % (w/w), or 0.01 to 0.5 % (w/w).

Preferred examples of the "cyclo-hispro salt" included in the feed additive of the present invention may include the types of the "pharmaceutically acceptable salt" and the "foodologically acceptable salt" described above.

As used herein, the term "subject" refers to a normal subject in need of improvement in exercise performance ability, improvement in muscle function, or enhancement of muscle strength, as well as a subject that has already developed or may develop a muscle disease, and the subject refers to all mammals including humans, dogs, cows, horses, rabbits, mice, rats, chickens, or humans, but the mammals of the present invention are not limited by the above examples. This term does not indicate a specific age or gender. Thus, it is intended to include adult/adult and newborn subjects, as well as fetuses, whether female/female or male/male. A patient refers to a subject suffering from a disease or disorder. The term patient includes human and veterinary subjects.

In one embodiment, the subject can be a human being aged at least about 50 years, at least about 55 years, at least about 60 years, or at least about 65 years. In another embodiment, the subject can exclude a human being aged at least about 50 years, at least about 55 years, at least about 60 years, or at least about 65 years.

In another embodiment, the subject may be a human patient with type 2 diabetes having excessive loss of skeletal muscle. In another embodiment, the subject may be a human patient with excessive loss of skeletal muscle but not having diabetes. In another embodiment, the subject may exclude a human patient with diabetes.

In another embodiment, the subject may be a human patient suffering from muscle dysfunction, muscle atrophy, muscle wasting or muscle degeneration due to a genetic mutation.

In another embodiment, the subject may be a patient who has received, is receiving, or will receive anticancer treatment, particularly chemotherapy.

In another embodiment, the subject is a subject in need of improved exercise performance, which may be in a state of poor or insufficient exercise performance relative to a particular training requirement. The subject in need of improved exercise performance may be experiencing symptoms of fatigue and/or muscle fatigue. The subject in need of improved exercise performance may be a subject engaged in physical exertion, particularly in an athletic event or competition.

In another embodiment, the subject may be a subject who desires to improve or enhance athletic performance. The subject who desires to enhance athletic performance may not be a subject who lacks or is not insufficient in athletic performance, but may be a subject who desires to increase or enhance athletic performance compared to his or her normal state. The subject who needs improved athletic performance may be a subject who is particularly interested in performing physical exertion in an athletic game or competition.

The optimal dosage and dosing interval for individual administration of cyclo-hispro or its salts will be determined by the nature and extent of the disease being treated, the dosage form, route and site of administration, and the age and health of the particular patient being treated, and it will be appreciated by those skilled in the art that the physician will ultimately determine the appropriate dosage to be used. Such administrations may be repeated as often as appropriate. If side effects occur, the dosage and frequency may be modified or reduced in accordance with routine clinical practice.

The route of administration of cyclo-hispro or a salt thereof may be administered via any common route as long as it can reach the target tissue. The cyclo-hispro or a salt thereof according to the present invention may be administered intraperitoneally, intravenously, subcutaneously, intradermally, or orally, depending on the purpose, but is not limited thereto. In addition, the cyclo-hispro or a salt thereof may be administered by any device capable of transporting to the target cell.

Hereinafter, the present invention will be described in more detail through examples. However, the present invention can be modified in various ways and can have various forms, and the specific examples and descriptions described below are only intended to help understand the present invention, and are not intended to limit the present invention to specific disclosed forms. It should be understood that the scope of the present invention includes all modifications, equivalents, or substitutes included in the spirit and technical scope of the present invention.

[Example 1]

Evaluation of muscle function improvement following CHP treatment

1-1. Breeding of laboratory animals and CHP administration

For experiments on older animals, 18-month-old C57BL6/J male mice were purchased from the Korea Basic Science Institute (KSBI). Mouse feed from Purina was purchased, and Cyclo-His-Pro (CHP) was purchased from Bachem.

Mice were housed in a constant temperature and humidity chamber maintained at 23±3℃, 50% humidity, and a 12-h light/dark cycle with free access to food and water. The mice were weighed and randomly distributed to have equal average weights and divided into two groups as shown in Table 1. The CHP administration group was administered 35 mg/kg of CHP orally once/day for 4 months. The control group was administered the same amount (200 μl) of distilled water.

group Control group CHP administration group Administration (oral) Vehicle (distilled water) CHP
(35 mg/kg) Marie Soo 7 7

1-2. Survival rate

As confirmed in Figure 1, the control group showed a survival rate of 71.4% with two mice dying of natural aging during the experimental period, but the CHP administration group showed normal vital signs and a survival rate of 100%.

1-3. Grip strength test

To measure the change in muscle strength due to CHP, a grip strength test was performed on animals in the control and CHP-administered groups. The maximum grip strength of the forepaws and hind paws that appeared when the tail of the mouse was held and the mouse was made to hold the grid of the grip strength test device and the tail was pulled backward was measured using a grip strength meter (Bioseb). The grip strength was measured repeatedly three times and the average value was used after being corrected for body weight. As shown in Fig. 2a, the maximum muscle strength of the CHP-administered group significantly increased by about 23.6% compared to the control group.

1-4. Four limb-wire hanging test

The limb hanging test is a muscle function measurement method that comprehensively measures muscle strength, coordination, and tolerance to fatigue in rodents, and is a method to measure continuous muscle function of mice against body weight (Lee et al., The Effects of 8-week Acetic Acid Feeding on Endurance Performance and Fat Metabolism in Skeletal Muscle of Mice, Exerc Sci, 2021). The mouse was placed on a 10x10 cm wire grid, carefully turned over, and the time until it fell to the floor was measured. The height to the floor was set to 40 cm, and a cushion was placed on the floor where the mouse fell to prevent shock caused by falling. The longest hanging time among the two measurements was recorded. As a result of the experiment, as shown in Figure 2b, the hanging time of the CHP administration group significantly increased by about 1.9 times compared to the control group.

1-5. Sensorimotor function evaluation (Rota-rod test)

Sensorimotor function assessment is a method to measure motor coordination and balance in animals (Robert M J Deacon, Measuring motor coordination in mice, J Vis Exp, 2013). Mice were placed on a rotating cylinder of a Rota-rod device (Harvard Apparatus) that rotated at 4 rpm, and the speed was steadily increased to 40 rpm for 5 minutes, and the time until they lost their balance and fell to the floor was measured. The experiment was conducted 3 times/day for a total of 3 days, and the average value of the 3 measurements was used. As shown in Fig. 2c, the CHP-administered group showed improved sensorimotor function compared to the control group, and statistical significance was confirmed on the 3rd day.

From the above results, it was found that CHP intake significantly improved muscle strength, motor coordination, resistance to muscle fatigue, and sense of balance.

[Example 2]

Changes in muscle weight and muscle fiber size following CHP treatment

2-1. Muscle weight measurement

When the mice of Example 1 reached 24 months of age, the mice were euthanized and the gastrocnemius (GM), tibialis anterior (TA), and cardiac muscle tissues were isolated. The weights of each tissue were measured and compared, and as shown in Fig. 3a, it was confirmed that the weight of the TA muscle significantly increased due to CHP administration.

2-2. Measurement of muscle fiber size improvement

Formalin-fixed GM tissues were sliced into paraffin sections and the muscle fibers were stained using H&E staining. As a result of measuring the cross-sectional area of each muscle fiber, as shown in Figures 3b, 3c, and 3d, it was confirmed that in the CHP-administered group, the distribution of muscle fibers over a wide area increased, and the average cross-sectional area of muscle fibers significantly increased by about 22% compared to the control group.

From the above results, it was found that CHP had a positive effect on changes in muscle fiber size as well as quantitative changes in muscles.

[Example 3]

Measurement of changes in muscle gene expression following CHP treatment

Muscle tissue was extracted RNA using NucleoZOL (MACHEREY-NAGEL) according to the manufacturer's total RNA isolation protocol, and 1 μg of RNA was reverse transcription polymerase chain reaction (RT-PCR) to synthesize cDNA using ReverTra Ace qPCR RT Master Mix (Toyobo). The synthesized cDNA was analyzed by real-time PCR using primer sets of myostatin, atrogin-1, foxo-1, dystrophin, sirt-1, sirt-3, and nrf-2 genes known to be related to aging and muscle atrophy and SYBR Green Realtime PCR Master mix (Toyobo) to quantify the expression level. The expression value of each gene was corrected by dividing it by the expression value of the housekeeping gene β-actin . Each primer set was synthesized by request from Bioneer, and the base sequence information is shown in Table 2.

Gene Forward (5'-3') Sequence number Reverse (5'-3') Sequence number Myostatin ACCCGTCAAGACTCCTACAA 1 CCTGGGCTCATGTCAAGTTT 2 Atrogin-1 TCAAAGGCCTCACGATCACC 3 TCAAACGCTTGCGAATCTGC 4 Foxo-1 CCTTTCCTCCTCCCTCTG 5 TGCCTCTACTGAATGATTACA 6 Dystrophin AAGAGGAAGAAATGCCCCCG 7 CCATGCGGGAATCAGGAGTT 8 Drp-1 AGGAGAAGAGGAAGCAAGCG 9 TAGGCTTTCCAGCACTGAGC 10 Err-α CAGGAGGCAGACACTGAT 11 CGGATTAAGCAGCAGCAA 12 Sirt-1 GTTGACCGATGGACTCCTCAC 13 GAGCTGGCGTGTGACGTTC 14 Sirt-3 ATGTCACTCACTACTTCCTG 15 ATCCCAGATGCTCTCTCA 16 Nrf-2 CAGCATAGAGCAGGACATGGAG 17 GAACAGCGGTAGTATCAGCCAG 18 Pgc-1α AAGGACTCTGAGAACACTTG 19 CAACTGACCCAAACACTTTAC 20 Cox-4 TACTTCGGTGTGCCTTCGA 21 TGACATGGGCCACATCAG 22 Tfb1m GGCTGAGAGACTTGTAGCCACT 23 AGGTGCACCACTCCTACATCAA 24 Tfb2m TTTGGCAAGTGGCCTGTGAC 25 ACTGATTCCCCGTGCTTTGACT 26 Murf-1 GCTACCTTCCTCTCAAGTGC 27 CTCAAGGCCTCTGCTATGTG 28 β-actin GGGAAGGTGACAGCATTG 29 ATGAAGTATTAAGGCGGAAGATT 30

As a result of the experiment, as shown in Fig. 4, it was confirmed that the expression of genes related to aging and muscle atrophy was maintained lower in the CHP administration group compared to the control group. In particular, sirt-1 , a representative anti-aging gene, was confirmed to increase equally in GM and TA muscles. Sirt-1 acts as a major regulator of energy and metabolic homeostasis and is known to be a major indicator reflecting the rate of cellular aging as it decreases with aging (Lagouge et al., Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha, Cell, 2006). Through this, it was found that CHP can suppress muscle aging and muscle atrophy. In addition, as the expression of the nrf-2 gene, which is at the core of the antioxidant action, was high in the CHP administration group, it was found that CHP also exhibited an antioxidant effect on muscles that increases resistance to oxidative stress.

Meanwhile, Sirt-1 is known to regulate pgc-1α, which is important for maintaining mitochondrial biogenesis and function (Handschin et al., Peroxisome Proliferator-Activated Receptor γ Coactivator 1 Coactivators, Energy Homeostasis, and Metabolism, Endocrine Reviews, 2006). In addition, the expression of Err-α, Cox-4, Drp-1, Tf1bm, and Tf2bm, genes related to mitochondrial biogenesis and function, was examined in GM and TA muscles. Likewise, the expression value of each gene was corrected by dividing it by the expression value of β-actin, a housekeeping gene. Each primer set was synthesized and used by Bioneer, and the base sequence information is shown in Table 2.

As a result of the experiment, as shown in Fig. 4, it was confirmed that the expression of related genes significantly increased by CHP administration.

From the above results, it was found that CHP improved muscle function by suppressing the expression of factors related to aging and muscle atrophy and promoting the expression of anti-aging and antioxidant genes including mitochondrial function.

[Example 4]

Measurement of citrate synthase activity in muscle

Citrate synthase is an enzyme that catalyzes citrate synthesis in the first step of the TCA cycle in mitochondria, and the activity level of this enzyme is used as a biomarker reflecting the amount of mitochondria in muscles (Vigelso et al., The relationship between skeletal muscle mitochondrial citrate synthase activity and whole body oxygen uptake adaptations in response to exercise training, Int J Physiol Pathophysiol Pharmacol, 2014.). To measure citrate synthase activity in muscles, proteins were extracted from TA muscles and reacted with oxaloacetate and acetyl-CoA at 37°C to generate CoA-SH. Subsequently, the absorbance was measured at 412 nm using the chromogenic reagent 5,5'-dithiose (2-nitrobenzoic acid), and the value was corrected for the total protein concentration.

As shown in Fig. 5, the experimental results confirmed that the citrate synthase activity of TA muscle significantly increased by about 12% compared to the control group due to CHP. This result means that the increase in the expression of genes related to mitochondrial biogenesis and function was reflected in the actual quantitative increase of mitochondria.

[Example 5]

Measurement of gene expression changes associated with myocardial atrophy

As a result of CHP reducing skeletal muscle aging and muscle atrophy and improving muscle function, changes in cardiac atrophy indices caused by aging were also investigated. As a result of measuring the expression of atrogin-1 and murf-1 genes, known as risk factors for cardiac atrophy, using the same method as in Example 3, it was confirmed that the expression of the two genes was significantly reduced by CHP.

From the above results, it was found that CHP very effectively improves muscle function by reducing risk factors for muscle atrophy in skeletal muscle as well as cardiac muscle, and inhibiting muscle aging by increasing antioxidant and mitochondrial activity.

Statistical Analysis

The statistical significance of the data in Figs. 1, 2a to 2c, 3a to 3d, 4, 5, and 6 was analyzed by the t-test statistical method. *p<0.05, **p<0.01, ***p<0.001.

[Example 6]

Confirmation of CHP effect in DMD animal model

6-1. Animal models and CHP treatment

C57BL/10ScSn (BL10) and C57BL/10ScSn-Dmdmdx (mdx) mice were used in this study. Mice were housed at 22°C and allowed free access to food and water. After one week of adaptation, mice were divided into three groups: CHP-treated mdx, water-treated mdx (control), and water-treated BL10 (control). Oral gavage was administered three times a week.

For the prophylactic protocol, mice were administered CHP or water (control) at a dose of 20 mg/kg from 3 to 20 weeks of age. For the therapeutic protocol, mice were administered CHP or water (control) at a dose of 35 mg/kg from 7 to 22 weeks of age. At the end of the study, mice were euthanized and tissues were collected. For biochemical analysis, tissues were collected, rapidly frozen, and stored at -80°C.

6-2. Evil Strength Test

Grip strength tests were performed at week 10 in the prevention protocol and week 16 in the treatment protocol, respectively, as follows: Grip strength of each mouse on each limb was measured on a pull-down grid assembly connected to a grip dynamometer (Columbus Instruments). Each individual mouse was pulled along a straight line parallel to the grid until the grip broke, providing maximum force (grams). This was repeated three times with a 5-minute interval between each measurement.

As a result, as confirmed in Figures 7a and 7b, the mdx mouse groups treated with water in the prevention protocol and the treatment protocol showed a significant decrease in limb muscle strength, but the mdx mouse group treated with CHP showed a significant increase in grip strength.

6-3. Hanging Test

The hanging test was performed at week 15 of the treatment protocol as follows. Mice were acclimated to the testing room 30 min before the experiment. The animals were placed on a wire grid and were allowed to grasp the grid with their limbs. The grid was then inverted so that the animals were hung upside down, and the time the mice could stay on the grid was measured. The maximum test length was 2 min 30 s (150 s). The latency to fall was measured five times for each mouse with 10 min intervals between trials.

As a result, as confirmed in Fig. 8, the average hanging time of the mdx mouse group treated with water significantly decreased, but the average hanging time of the mdx mouse group treated with CHP significantly increased.

The above results confirmed that CHP has the effect of increasing limb muscle strength and exercise endurance.

[Example 7]

Contractile and force-generating effects of CHP

7-1. Ex vivo muscle strength assessment

Force production in limb muscles was tested ex vivo in extensor digitorum longus (EDL) and soleus muscles isolated from mice treated with CHP for 17 weeks.

Muscle mechanical measurements were assessed according to the method described in the literature [N. Zanou et al. , "Role of TRPC1 channel in skeletal muscle function," Am. J. Physiol. Cell Physiol. , vol. 298, no. 1, Jan. 2010] with minor modifications. All analyses were performed in a blinded manner. BL10, mdx, and CHP-treated mdx mice were euthanized by cervical dislocation. EDL and soleus muscles were rapidly dissected and immersed in a 10 mL horizontal chamber containing continuously oxygenated Krebs solution (25°C, pH 7.4) consisting of 135.5 mM NaCl, 5.9 mM KCl, 1 mM MgCl ₂ , 2 mM CaCl ₂ , 11.6 mM sodium HEPES, and 11.5 mM glucose. The muscle was strapped between a dual-mode lever arm and a fixed hook, and stimulation was delivered via platinum electrodes (1500A Intact Muscle Test System, Aurora Scientific Inc., Canada) running parallel to the muscle. Resting muscle length (L0) was carefully calibrated for maximal isometric force using 125 Hz maximal fusion tetani. The force-frequency relationship was determined by sequentially stimulating the muscle at stimulation streams of 25, 50, 75, 100, 125, and 150 Hz of 300 ms duration, with 1 min of rest between each contraction. Normalized muscle force (mN/mm ² ) was expressed as the cross-sectional area (CSA) obtained by dividing the muscle blot weight (mg) by the length and considering the fiber length as 0.5 L0 for the EDL and 1 for the soleus. Data from each experiment were analyzed using Aurora's DMA software (Aurora Scientific Inc., 2002; Solwood Enterprises, Inc., 2002) and Microsoft Excel.

7-2. Eccentric contraction of EDL muscle ex vivo

Eccentric contractions were performed as described in the literature [N. Zanou, Y. Iwata, O. Schakman, J. Lebacq, S. Wakabayashi, and P. Gailly, "Essential role of TRPV2 ion channels in the sensitivity of dystrophic muscle to eccentric contractions," FEBS Lett. , vol. 583, no. 22, pp. 3600-3604, Nov. 2009]. Briefly, the EDL muscle underwent a series of seven eccentric contractions consisting of a 1-mm stretch applied 160 ms after stimulus onset, followed by a 500-ms tonic twitch that lasted up to 250 ms after stimulus onset (with a 10-s interval between two consecutive tetani). Isometric force was measured for each tetanus immediately before stretch onset, and the percentage force decline relative to the first tetanus was calculated.

When tested for concentric contractions, the muscles of mdx mice showed overall lower force production and a downward shift in the force-frequency relationship compared to BL10 mice, as shown in Figs. 9a to 9f. The performance of EDL and soleus muscles was improved by CHP, with a partial recovery of maximal contractile force. In fact, this is consistent with the preservation of muscle strength observed in vivo. These results suggest that CHP treatment maintains force production.

[Example 8]

Ca2 ⁺ release/absorption recovery effect of CHP

There is increasing evidence that sarcolemma damage in DMD muscle fibers as a result of DAPC instability also contributes to the disruption of Ca2 ⁺ homeostasis. Muscle fiber Ca2 ⁺ influx and release is impaired in DMD patients, and the mdx model reports a similar phenotype. To evaluate the effect of CHP on Ca2 ⁺ dysregulation, flexor digitorum brevis (FDB) muscles were isolated from CHP-treated mice at the end of the prophylactic protocol as well as from healthy control and mdx control mice. Ca2 ⁺ release from the sarcoplasmic reticulum (SR) was monitored using the cytosolic Ca2 ⁺ indicator Fluo-4/AM in isolated fibers treated ex vivo with caffeine to stimulate Ca2+ release. The experimental procedures are described in more detail below.

FDB muscle fibers were loaded with the cytosolic Ca2 ⁺ indicator Fluo-4/AM (5 μM; Invitrogen, Basel, Switzerland) dissolved in Krebs Ca2 ⁺ solution [in mM: NaCl 135.5, _MgCl2 1.2, KCl 5.9, glucose 11.5, HEPES 11.5, CaCl2 1.8 (pH 7.3)] for 20 min in an incubator and then rinsed twice with Krebs solution.

For caffeine stimulation, fibers were washed twice and stored in Krebs Ca2 ⁺ solution. Fluo-4 fluorescence was monitored using a confocal microscope system (Zeiss LSM 5 Live, 40x oil immersion lens, excitation wavelength 488 nm and emitted fluorescence recorded between 495–525 nm) in a time-lapse acquisition framework. After recording basal fluorescence, fibers were stimulated with a final concentration of 2.5 mM caffeine (O1728-500, Thermofisher Scientific) to induce Ca2 ⁺ release from the SR. For store-operated ^{Ca2+ entry} (SOCE) measurements, fibers were washed twice in Ca2 ⁺ -free Krebs solution and then stored in Ca2 ⁺ -free Krebs solution immediately prior to image acquisition.

After recording baseline fluorescence, fibers were stimulated with a final concentration of 1 μM thapsigargin (Tg; T9033, Thermofisher Scientific) to evoke Ca2 ⁺ release from the SR. 2 mM _CaCl2 was finally used to assess SOCE. Zen software (products/microscopy-software/zenlite/zen-2-lite) was used for acquisition and data were exported to an Excel file for analysis. The use of the single excitation/release dye fluo-4 necessitates normalization to pre-stimulation values to account for possible differences in dye loading. The amplitude of SR Ca2 ⁺ stores was calculated by subtracting peak fluorescence from baseline. The amplitude of Ca2 ⁺ transients plateau induced by caffeine stimulation was calculated and expressed as a percentage of the SR Ca2 ⁺ peak amplitude, indirectly reflecting the contribution to SOCE. Actual SOCE assessed by Tg stimulation was calculated as the difference between the calcium amplitude after addition of 2 mM CaCl ₂ and the lowest Ca ²⁺ level before addition of CaCl ₂ . The lowest Ca ²⁺ level before CaCl ₂ addition was used to estimate the level of Ca ²⁺ reuptake (calculated as a percentage of the SR Ca ²⁺ peak).

As confirmed in Fig. 10a, the response to caffeine-induced Ca2 ⁺ release in the SR was impaired in mdx mice because mdx fibers undergo persistent depletion of intracellular Ca2 ⁺ stores, but was restored by CHP. As confirmed in Fig. 10b, Ca2 ⁺ uptake via store-operated calcium channels (SOCs) after SR depletion is normally upregulated in mdx as a compensatory mechanism for Ca2 ⁺ depletion, but CHP normalized Ca2 ⁺ uptake, indicating that Ca2 ⁺ handling was properly maintained. To directly quantify SOCE, stimulation of SR Ca2 ⁺ release was performed using thapsigargin instead of caffeine, which showed a similar trend of CHP-induced normalization, as confirmed in Fig. 10c and 10d.

Statistical Analysis

Statistical significance for the data in Figures 7a to 7b, 8, 9a to 9f, and 10a to 10d was analyzed by one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Claims (21)

A pharmaceutical composition for enhancing exercise performance comprising cyclo-hispro or a pharmaceutically acceptable salt thereof. In the first paragraph, a pharmaceutical composition exhibiting any one or more of the following effects: i) to v): i) Increased muscle strength; ii) Increased motor coordination; iii) Increased tolerance to muscle fatigue; iv) increased sense of balance; and v) Increased muscle energy production. A health functional food composition for improving exercise performance comprising cyclo-hispro or a food-chemically acceptable salt thereof. In the third paragraph, a health functional food composition exhibiting one or more of the following effects: i) to v): i) Increased muscle strength; ii) Increased motor coordination; iii) Increased tolerance to muscle fatigue; iv) increased sense of balance; and v) Increased muscle energy production. A feed additive for improving exercise performance comprising cyclo-hispro or a salt thereof. A pharmaceutical composition for improving muscle function or preventing or treating muscle disease, comprising cyclo-hispro or a pharmaceutically acceptable salt thereof. A pharmaceutical composition according to claim 6, wherein the muscle disease is a muscle disease caused by muscle dysfunction, muscle atrophy, muscle wasting or muscle degeneration. A pharmaceutical composition in claim 6, wherein the muscle disease is at least one selected from the group consisting of atony, muscular atrophy, muscular dystrophy, myasthenia, cachexia, sarcopenia, myocardia, and acardiotrophy. In the sixth paragraph, a pharmaceutical composition exhibiting any one or more of the following effects: i) to v): i) Increased muscle mass; ii) Increase in muscle fiber size; iii) Inhibition of muscle wasting; iv) inhibition of muscle wasting; and v) Increased muscle energy production. A health functional food composition comprising cyclo-hispro or a food-chemically acceptable salt thereof for improving muscle function or preventing or improving muscle disease. A health functional food composition in claim 10, wherein the muscle disease is a muscle disease caused by muscle dysfunction, muscle atrophy, muscle wasting, or muscle degeneration. A health functional food composition in claim 10, wherein the muscle disease is at least one selected from the group consisting of atony, muscular atrophy, muscular dystrophy, myasthenia, cachexia, sarcopenia, myocardia, and acardiotrophy. In the 10th paragraph, a health functional food composition exhibiting any one or more of the following effects: i) to v): i) Increased muscle mass; ii) Increase in muscle fiber size; iii) Inhibition of muscle wasting; iv) inhibition of muscle wasting; and v) Increased muscle energy production. A feed additive for improving muscle function or preventing or improving muscle disease, comprising cyclo-hispro or a salt thereof. A composition for strengthening muscle, comprising cyclo-hispro or a salt thereof. A method for improving exercise performance, comprising administering to a subject in need thereof an effective amount of cyclo-hispro or a pharmaceutically or food-wise acceptable salt thereof. A method for improving muscle function or preventing, improving or treating muscle disease, comprising administering to a subject in need thereof an effective amount of cyclo-hispro or a pharmaceutically or food-wise acceptable salt thereof. A method for strengthening muscle, comprising administering to a subject in need thereof an effective amount of cyclo-hispro or a pharmaceutically or food-wise acceptable salt thereof. Use of cyclo-hispro or a pharmaceutically or food-wise acceptable salt thereof for the manufacture of a drug or health functional food for improving exercise performance. Use of cyclo-hispro or a pharmaceutically or food-wise acceptable salt thereof for the manufacture of a medicament or health functional food for improving muscle function or preventing, improving or treating muscle diseases. Use of cyclo-hispro or a pharmaceutically or food-wise acceptable salt thereof for the manufacture of a muscle-enhancing drug or health functional food.

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